M80 light microscope

Individual fractions (n = 60) (Section 3.3) were tested for their anthelmintic effect on larvae (xL3s) of H. contortus using an established bioassay [11 (link)]. The assay was performed in triplicate. In brief, fractions in 40 μL of LB* (2000 μge/μL) were dispensed into the wells of sterile 368-well flat-bottom microtitre plates (cat. no. 3680; Corning, Corning, NY, USA) containing 80 xL3s; quadruplicate wells with no compound (LB* + 0.6% DMSO; negative control) or monepantel (Zolvix; Elanco, Greenfield, IN, USA), moxidectin (Cydectin; Virbac, Carros, France), monepantel/abamectin (Zolvix Plus; Elanco, Greenfield, IN, USA) and compound MIPS-0018666 (abbreviated here as M-666; ref. [28 (link)]) as positive-controls (20 µM). The motility of xL3s was measured at 90 h, and the development and phenotypic alterations of xL3s at 168 h. At 168 h, larvae in individual wells were fixed with 40 µL of 1% iodine and microscopically examined using a M80 light microscope (Leica, Wetzlar, Germany) at 60-times magnification to assess their development based on the presence or absence of a well-developed pharynx [27 (link)], as well as their morphology (phenotype) [7 (link),11 (link)]. At 168 h, xL3s exposed to LB* with ≤0.6% DMSO are expected to reach the L4 stage in vitro within 168 h [10 (link)].

Mice were time mated for two-four nights, with females plug checked daily for copulation plugs. Positive plugs were noted as day E0.5 and all females for which a plug was discovered were immediately separated from the male. Gestational length was measured in days post copulation by recording the morning of a copulation plug was detected as E0.5 and visually monitoring females twice daily (morning and afternoon) for births from late gestation (E18.5) until pups were delivered (E19.5-E21.5, depending on oocyte genotype). Pregnant females were euthanised and embryos were collected at E9.5, E12.5 E14.5, E17.5, and E18.5. E9.5 whole sacs were weighed, fixed in 4% PFA for 72 hr at 4 °C, processed, paraffin-embedded, and sectioned at 5 µm. Whole-mount images of E9.5 embryos were taken using a LEICA M80 light microscope with LEICA MC170 HD camera attachment. Embryos and placentas were collected from the same offspring at E12.5, E14.5, E17.5, and E18.5 and weighed separately. Placentas were then bisected, and half was fixed in 4% PFA for 72 hr at 4 °C, processed, and paraffin-embedded with the cut side of the placenta facing the front of the block. The other half of the placenta was rinsed in PBS, snap-frozen on dry ice, and stored at –80 °C for RNA analysis and RRBS. P0 (day of birth) or P3 pups were weighed, euthanised by decapitation, and samples collected as required.